Eucaryotic cells undergo dynamic cellular changes during cell division and differentiation. The manner in which cells coordinate different cellular processes during these times is poorly understood. The yeast Hsl1 (Nim1-related) protein kinase pathway has been implicated in a variety of cellular processes including monitoring the organization of the septin cytoskeleton, nuclear orientation, nuclear division, and cytokinesis. We have previously found that the septins can activate Hsl1 in vitro and in vivo and have identified several novel cellular processes in which Hsll participates. We propose to further determine how the Hsl1 is regulated and identify molecular targets of the kinase. To understand how septins interact with and activate Hsl1, we will further delineate the septin binding regions of Hsl1 and also determine the regions of the septins that bind to Hsll. We will also determine which septins bind to the related kinases Gin4 and Kcc4. Finally, we will determine if septin binding regulates the interaction of the Hsl7 with Hsl1. To further understand how Hs11 functions we will analyze proteins that interact with Hs11 physically and genetically. We have screened a yeast proteome microarray with a labeled fragment of Hsl1 and identified a number of candidate interacting proteins. Additional interacting proteins will be sought and each "interactor" will be tested for their roles as potential regulators or targets of Hsl1. We will also characterize CYK3, which was identified as a high copy suppressor of an hsl1delta mutation. CYK3 encodes a kinase implicated in cytokinesis. We will attempt to determine the relationship between Cyk3 and Hsl1 by determining if they function in the same pathway and if Hsl1 phosphorylates Cyk3. Finally, we will screen a yeast proteome array for substrates of Hsl1, Gin4 and Kcc4. Candidate in vitro substrates will be examined for phosphorylation in vivo using several criteria. The role of Hsl1 phosphorylation will be determined by mutating the sites in vivo and examining the effect on cells. Since the Hsl1 signaling pathway is highly conserved with other eukaryotes these studies are expected to provide general insight into how this pathway coordinates diverse cellular processes.